UC-NRLF 


b  ^  mi  fl?4 

STANFORD    UNIVERSITY    PUBLICATIONS 
UNIVERSITY    SERIES 

MEDICAL    SCIENCES 

Volume  I  Number  2 


A  Cytological  Study  of  the  Kidney  Cell 

in  Long  Continued  Hyperfixnction  with 

Relation  to  Hypertrophy  and  the 

Mitochondrial  Apparatus 


BY 


LUDWIG  A.  EMGE 
Assistant  Professor  of  Obstetrics  and  Gynecology 


From  the  Division  of  Obstetrics  and  Gynecology 

Stanford  University  Medical  School 

San  Francisco 


STANFORD  UNIVERSITY,  CALIFORNIA 

PUBLISHED  BY  THE  UNIVERSITY 

1921 


0^872. 
E-5-3 


MEMCAL    .SCHOOL 
LimAKY 


STANFORD    UNIVERSITY    PUBLICATIONS 
UNIVERSITY    SERIES 

MEDICAL    SCIENCES 

Volume  I  Number  2 


A  Cytological  Study  of  the  Kidney  Cell 

in  Long  Continued  Hyperfunction  with 

Relation  to  Hypertrophy  and  the 

Mitochondrial  Apparatus 


BY     / 

LUDWIG  A.  EMGE 
Assistant  Professor  of  Obstetrics  and  Gynecology 


From  the  Division  of  Obstetrics  and  Gynecology 

Stanford  University  Medical  School 

San  Francisco 


STANFORD  UNIVERSITY,  CALIFORNIA 

PUBLISHED  BY  THE  UNIVERSITY 

1921 

n 


Stanford  University 
Press 


\©Z-\ 


CONTENTS 

PAGE 

Introduction 107 

Technique 110 

Results  of  Vital  Staining  Studies Ill 

Results  of  Fixed  Tissue  Studies 114 

Summary           120 

Bibliography 121 

Explanation  of  Plates 123 

Plates          125 


9563 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

Microsoft  Corporation 


http://www.archive.org/details/cytologicalstudyOOemgerich 


A  Cytological  Study  of  the  Kidney  Cell  in  Long  Continued 

Hyperfunction  with  Relation  to  Hypertrophy  and 

the  Mitochondrial  Apparatus 

Ever  since  the  knowledge  of  mitochondria  had  passed  the  mere 
descriptive  stage  numerous  attempts  have  been  made  to  interpret  their 
physiological  significance.  This  investigation  was  undertaken  in  order  to 
determine  whether  wide  variations  in  the  duration  and  degree  of  experi- 
mentally induced  renal  activity  were  associated  with  any  demonstrable 
peculiarities  in  the  mitochondrial  content  and  general  cellular  structure  of 
the  renal  tubules.  Part  of  the  tissues  for  this  study  were  obtained  through 
the  courtesy  of  Dr.  Addis  and  Mr.  Shevky  of  the  Division  of  Medicine, 
who  have  kindly  contributed  the  life  history  of  the  animals  used. 

NOTE  BY  T.  ADDIS  AND  A.  E.  SHEVKY  ON  THE  LIFE  HISTORY  OF 

THE  MATERIAL  DEALT  WITH 

(From  the  Division  of  Medicine) 

There  is  no  direct  method  by  which  the  "work"  of  the  kidney  can  be  measured, 
but  changes  in  rate  of  work  may  be  assumed  to  be  indicated  by  changes  in  rate 
of  metabolism.  Barcroft(l)*  has  shown  that  an  increased  rate  of  urea  excretion 
is  associated  with  an  increased  consumption  of  oxygen  by  the  kidney,  although 
increases  in  sodium  chloride  and  water  excretion  are  not  accompanied  by  any 
appreciable  change  in  gaseous  metabolism.  On  these  grounds  one  would  expect  to 
be  able  to  produce  renal  hypertrophy  in  intact  animals  by  increasing  the  work  of 
their  kidney  by  constantly  and  over  a  long  period  giving  them  large  amounts  of 
urea  in  their  food.  At  least  such  a  result  might  be  expected  if  the  assumption  is 
correct  that  the  increase  in  size  of  the  remaining  kidney  after  unilateral  nephrectomy 
is  due  to  the  additional  work  it  has  to  perform. 

Twenty-four  newly-weaned  white  rats  about  a  month  old  were  divided  into 
two  groups  as  nearly  as  possible  alike  in  regard  to  the  size,  weight,  and  ancestry 
of  the  individuals  of  which  they  were  composed.  Each  group  was  kept  in  a  separate 
cage  of  the  type  described  by  Robertson (2).  The  diet  used  was  one  shown  by 
Osborne  and  Mendel  (3)  to  be  adequate  for  growth  and  maintenance.  It  contained 
15  gms.  of  lard,  33  gms.  of  cornstarch,  5  gms.  of  agar-agar,  and  43.5  gms.  of 
powdered  milk.  In  order  to  facilitate  the  combination  of  these  ingredients  into  a 
homogeneous  paste  into  which  urea  could  be  easily  incorporated  we  added  50  cc. 
of  whole  milk  for  every  100  gms.  of  the  combined  foods.  Urea  in  substance  was 
rubbed  in  a  mortar  into  that  part  of  the  food  given  to  the  urea-fed  rats,  and  in 
order  to  obviate  any  possibility  of  error  a  little  charcoal  was  added  at  the  same 
time.  For  the  first  two  months  5.5  gms.  was"  given  with  each  100  gms.  of  food, 
during  the  third  month  8  gms.,  for  the  fourth  and  fifth  months  11  gms.,  and  so  on 
with  successive  increases  until  from  the  13th  to  the  16th  month,  when  the  experi- 
ment was  terminated,  17  gms.  of  urea  was  taken  in  every  100  gms.  of  food.   There 


*  Figures  in  parenthesis  refer  to  bibliography  on  pages  121  and  122. 


108 


A  CYTOLOGICAL  STUDY  OF  THE  KIDNEY  CELL 


was  no  limitation  of  water  intake,  except  that  with  the  idea  of  straining  the  urea- 
concentrating  capacity  of  the  kidney,  we  removed  the  water  tube  from  the  cage  of 
the  urea-fed  rats  for  one  hour  each  day  following  the  giving  of  fresh  food. 

There  can  be  no  doubt  that  by  these  measures  we  succeeded  in  making  the 
kidneys  of  the  urea-fed  group  do  much  more  work  than  those  of  the  controls.  In 
spite  of  repeated  efforts,  however,  we  cannot  give  any  accurate  measure  of  the 
difference,  since  we  did  not  succeed  in  entirely  overcoming  the  technical  difficulties 
of  collecting  urine  from  such  small  animals.  We  did  find,  however,  that  the  urea- 
fed  rats  topk  about  twice  as  much  water  and  excreted  about  twice  as  much  urine. 
This  urine  at  times  contained  as  much  as  10  per  cent  of  urea.  The  funnel  over 
which  the  urine  ran  into  the  collecting  vessel  was  usually  coated  with  urea  crystals. 
Examination  of  the  feces  showed  that  urea  was  not  excreted  by  the  gastro- 
intestinal tract. 

At  the  end  of  sixteen  months  various  accidents  had  reduced  the  number  of 
rats  to  eight,  four  controls  and  four  urea-fed.  After  a  functional  test,  which  will 
be  described  below,  these  rats  were  killed  by  bleeding  from  the  carotid  artery  after 
very  short  ether  anaesthesia,  and  the  kidneys  at  once  removed  and  weighed.  The 
results  are  given  in  Table  I.    They  fail  to  show  the  existence  of  any  hypertrophy. 


TABLE  I 
KIDNEY  WEIGHTS  OF  CONTROL  AND  UREA-FED  RATS 

Controls 

Kidney  Weight  as  a 

Percentage  of  Body 

Weight-Alimentary  Tract 

per  cent. 

0.75 

0.75 

0.85 

0.99 


No.       Kidney  Weight       Body  Weight- Alimentary  Tract 


gms. 
1.37 
0.93 
1.13 
1.30 


gms. 
182 
124 
132 
132 


No. 

5 
6 
7 
8 


Kidney  Weight 
gms. 
1.27 
1.01 
1.40 
1.06 


Urea -Fed 


Body  Weight-Alimentary  Tract 
gms. 
148 
115 
152 
103 


Kidney  Weight  as  a 

Percentage  of  Body 

Weight-Alimentary  Tract 

per  cent. 

0.86 

0.89 

0.93 

0.97 


On  the  day  before  each  animal  was  killed  an  attempt  was  made  to  measure 
the  urea-excreting  capacity  of  the  kidneys.  In  order  that  both  groups  might  be 
under  the  same  conditions,  no  urea  was  given  to  the  urea  group  for  two  days 
before  the  experiment.  Urea  in  concentrated  solution,  0.5  gms.  for  one  pair  of 
rats,  and  0.375  for  the  other  three  pairs,  was  injected  from  a  syringe  through  a 
fine  tube  into  the  stomach.  The  rat  was  then  placed  over  a  funnel,  so  that  all 
urine  was  collected.  After  three  hours  the  carotids  were  cut  under  ether  anaes- 
thesia, and  the  blood  collected.  The  urea  content  of  both  blood  and  urine  was 
determined  by  the  usual  urease  aeration  method.    The  results  are  given  in  Table  II. 


INTRODUCTION 


109 


TABLE  II 
UREA-EXCRETING   FUNCTION    OF   CONTROL   AND   UREA-FED    RATS 

Controls 

Ratio:  Urea  in  Urine 
Urea  in  Blood 


0.45 
0.26 
0.34 
0.06 


Mo. 

Urea  in  One  Hour's 

Urea  in  lOOcc 

Urine 

Blood 

mgs. 

mgs. 

1 

75 

165 

2 

48 

186 

3 

34 

76 

4 

8 

132 

Urea-Fed 

»o. 

Urea  in  One  Hour's 

Urea  in  lOOcc 

Urine 

Blood 

mgs. 

mgs. 

5 

85 

102 

6 

44 

93 

7 

64 

140 

8 

58 

60 

Ratio :  Urea  in  Urine 
Urea  in  Blood 

0.83 
0.47 
0.46 
0.97 


The  higher  rate  of  urea  excretion  in  spite  of  the  lower  level  of  blood  urea 
concentration  in  the  urea-fed  rats  seems  to  indicate  a  greater  facility  in  the  excre- 
tion of  urea,  in  spite  of  the  absence  of  any  appreciable  increase  in  the  size  of  their 
kidneys.  But  the  number  of  experiments  is  much  too  small  to  allow  for  any 
definite  conclusion. 

The  two  groups  of  animals  whose  physiological  history  has  been  de- 
scribed by  Addis  and  Shevky  were  supplemented  with  another  two  groups 
of  white  rats  similar  in  age  and  weight.  One  group  was  fed  general  food 
and  the  other  was  given  urea  food  as  noted  in  the  life  history  of  the  tissues 
of  the  first  two  groups.  After  several  days'  feeding,  the  first  pair,  urea- 
fed  and  control,  was  killed.  Then  within  two  weeks  a  pair  was  killed 
every  other  day.  The  idea  was  to  obtain  tissues  for  study  of  possible 
early  changes  in  the  renal  cellular  structure  produced  by  urea  feeding  for 
a  comparison  with  tissues  from  kidneys  in  a  normal  state  of  activity. 

There  were,  therefore,  four  distinct  groups  available  for  comparative 
study;  the  first,  composed  of  kidneys  which  since  early  youth  had  been 
subjected  to  a  constant  strain  in  the  excretion  of  large  amounts  of  urea; 
the  second,  a  group  which  had  been  fed  a  similar  diet  without  urea  but 
had  been  given  a  single  dose  of  urea  just  before  death ;  the  third,  a  group 
which  was  fed  urea  over  a  period  of  from  two  to  fourteen  days  before 
death ;  and  the  fourth,  a  group  fed  on  ordinary  diet  which  received  no 
urea  whatsoever. 


110  a  cytological  study  of  the  kidney  cell 

Technique 

Owing  to  the  fact  that  the  kidneys  had  to  be  weighed  for  reasons 
explained  in  the  above  life  history,  fixation  by  arterial  injection  could  not 
be  practiced.  This  would  in  any  case  have  been  impracticable  since  it 
would  not  have  permitted  vital  studies. 

As  fixing  agents  Bensley's(4)  formalin-zenker,  Regaud's(5)  neutral 
formalin-bichromate,  and  Kolster's(6)  chromalum  and  fluorchrome  mix- 
tures were  used.  Controls  were  also  fixed  in  chemically  pure,  freshly  neu- 
tralized 10  per  cent  formaldehyde.  It  was  impossible  to  obtain  osmic  acid 
at  the  time  of  this  investigation  (1918).  Tissues  in  pieces  of  2  mm.  thick- 
ness were  fixed  in  these  agents  from  one  to  three  days  and  then  chromi- 
cized  from  three  to  nine  days.  A  temperature  of  about  body  heat  was 
maintained  throughout  the  period  of  fixation.  The  fluids  were  changed 
daily.  The  fixed  material  was  cleared  with  either  cedar  oil,  bergamot  oil, 
or  xylol.  I  have  found  practically  no  difference  in  the  shrinkage  of  the 
tissues  with  either  of  the  three,  and  therefore  have  used  xylol  in  prefer- 
ence as  it  is  the  fastest  and  least  expensive  of  all  clearing  agents.  Paraf- 
fin, 60°,  was  used  as  the  embedding  medium.  Sections  were  cut  serially 
and  about  5  \i  thick.  Altmann's(7)  anilin-fuchsin  picric  acid  method  in 
its  original  form  and  as  modified  by  Cowdry(8)  and  Galeotti(9),  Bens- 
ley's  (4)  anilin-acid-fuchsin  methyl  green,  Bensley's(4)  copper-chrome- 
hematoxylin  and  Heidenhain's  iron-hematoxylin  were  used.  Simple  con- 
ventional stains  were  employed  as  a  check  on  the  general  picture.  Ap- 
parently it  is  not  necessary  to  use  osmic  acid  to  obtain  good  fuchsin  pic- 
tures. Such  simple  bichromate  mixtures  as  mentioned  above  will  pre- 
serve mitochondria  excellently  and  allow  good  staining  results. 

Among  the  fixatives  the  formalin-zenker  and  the  neutral  formalin 
bichromate  mixtures  with  a  period  of  chromization  have  given  by  far  the 
best  results.  Of  the  staining  methods  the  anilin-acid-fuchsin  methods 
are  quicker  but  otherwise  in  no  way  superior  to  the  other  methods 
mentioned. 

Fresh  tissues  were  studied  in  a  normal  saline  solution  of  1 :  10,000 
Janus  green  p\  Arnold's (10)  neutral  red  and  methylene  blue  were  used 
in  several  control  animals.  Janus  green  studies  of  renal  tissues  are  very 
difficult.  First  of  all,  the  dye  seems  to  penetrate  very  slowly,  and  one  is 
therefore  not  certain  whether  the  mitochondria  have  not  changed  in  ap- 
pearance by  the  time  they  stand  out  plainly ;  secondly,  mitochondria  are 
usually  massed  so  densely  in  the  convoluted  tubules  that  a  clear  picture  is 
very  rarely  obtained ;  thirdly,  in  order  to  obtain  thin  enough  pieces  of 
tissue  one  must  either  tease  them  out  or  cut  them  with  the  freezing  micro- 
tome. Either  method  furnishes  ground  for  controversy.  It  seemed  to 
me,  however,  that  sections  cut  with  a  good-  freezing  outfit  are  preferable 
to  teased-out  tissues. 


results  of  vital  staining  studies  111 

Results  of  Vital  Staining  Studies 
Nothing  remarkable  was  found  in  the  vital  staining  studies.  It 
was  at  once  plain  that  in  spite  of  the  poor  penetration  of  the  dye,  the  mito- 
chondria of  the  convoluted  apparatus  by  far  outnumbered  those  of  the 
remaining  tubular  structure.  Comparing  controls  to  urea-fed  rats  of  the 
first  group,  there  was  no  striking  difference  in  the  mitochondrial  picture 
in  general.  In  all  tissues  studied  the  impression  was  gained  that  in  the 
control  rats  the  mitochondria  of  the  convoluted  tubes  were  arranged  in 
distinct,  closely  packed  stout  rods  or  in  bands  of  medium-size  granules, 
which  extended  about  two-thirds  into  the  cell  and  which  were  most  nu- 
merous near  the  base.  In  the  collecting  apparatus  the  finer,  less  numerous 
granular  mitochondria  were  distributed  throughout  the  cell,  leaving  a 
clear  space  around  the  nucleus.  In  the  large  tubules  of  Bellini  the  stout 
rodlike  form  appeared  again,  but  the  number  was  few,  and  I  judged  that 
more  than  half  of  the  cells  were  mitochondria-free.  In  the  urea-fed  rats 
the  only  difference  in  the  mitochondrial  picture  was  that  the  granular 
form  in  general  prevailed,  but  position  and  arrangement  seemed  to  be  the 
same  as  in  the  controls.  At  any  rate,  no  such  morphological  differences 
existed  as  would  warrant  inferences  as  to  differences  in  secretory  activity 
in  the  proximal  convoluted  tubules  of  the  four  groups  of  kidneys. 

The  use  of  vital  color  reactions  to  solve  the  secretory  problem  of  the 
kidneys  dates  back  to  1874,  when  Heidenhain  ( 1 1 )  and  Neisser  first  in- 
jected dyestuffs  into  animals  and  found  them  in  the  convoluted  tubules, 
while  the  glomeruli  and  capsules  were  free  from  color  materials.  This 
work  in  general  has  been  substantiated  by  a  number  of  investigators. 
Since  the  exhaustive  histochemical  studies  of  Leschke(12),  confirmed  by 
OHver(13),  it  seems  more  clear  than  ever  that  urea  and  perhaps  other 
urinary  solids  are  secreted  (or  excreted)  in  the  convoluted  apparatus, 
while  the  glomerular  apparatus  is  primarily  concerned  with  the  secretion 
of  practically  solid-free  fluids.  The  difficulty  enters  when  one  tries  to 
obtain  evidence  by  vital  dye  studies  as  to  whether  the  mitochondrial  ap- 
paratus is  directly  concerned  with  the  secretory  mechanism  of  the  cell 
of  the  convoluted  tubule.  One  must  not  forget  that  most  of  the  dyestuffs 
used  in  histological  methods  are  of  colloidal  nature,  and  consequently 
when  seen  in  cells  that  secrete  substances  crystalloid  in  nature  they  cannot 
be  used  as  an  absolute  criterion  or  parallel.  The  presence  of  dye  mate- 
rials in  the  renal  cells  brought  there  by  the  blood  or  lymph  stream  must 
be  considered  a  physical  rather  than  a  chemical  reaction,  as  was  brought 
out  by  Suzuki(14)  working  in  Aschoff's  laboratories.  He  found  that  the 
dye  appeared  in  the  urine  long  before  the  granular  structures  of  the  con- 
voluted tubules,  showed  any  signs  of  staining.  Carmine  in  various,  con- 
centrations was  used  and  at  an  "early  stage  of  excretion"  many  finely 
granular  carmine  casts  were  present  in  the  collecting  tubules.     Suzuki 


112  A  CYTOLOGICAL  STUDY  OF  THE  KIDNEY  CELL 

states  that  the  presence  of  these  granules  does  not  point  to  carmine- 
stained  secretory  granules  derived  from  the  epithelium  of  the  convoluted 
tubules,  but  rather  to  a  product  of  precipitation  of  the  dyestuff  in  solu- 
tion, which  is  only  in  part  passed  through  the  convoluted  tubules,  while 
some  of  it  passes  through  the  glomerular  apparatus.  He  calls  attention  to 
the  fact  that  by  pushing  the  carmine  injections  there  is  a  gradual  accumu- 
lation of  dyestuff  in  the  renal  epithelium  which  ultimately  leads  to  stain- 
ing of  the  true  cell  granules.  The  latter,  therefore,  must  be  sharply  differ- 
entiated from  carmine  granules.  His  results  lead  him  to  believe  that  the 
carmine-absorbing  granules  have  nothing  to  do  with  the  actual  secretion 
of  the  dye  material. 

The  objection  that  might  be  raised  is  that  carmine  is  not  a  specific 
vital  dye  for  mitochondria.  Nevertheless  the  fundamental  result  remains 
unchanged.  Many  investigators  are  in  accord  that  the  striations  of  the 
convoluted  tubules  of  the  kidney  are  homogeneous  rods  when  seen  in 
fresh  tissues,  but  become  granular  in  appearance  under  various  condi- 
tions. Some  have  considered  this  a  true  secretory  phenomenon,  others  an 
artefact.  Arnold(15)  thinks  that  the  individual  granules  of  the  living 
renal  epithelium  are  pre-existing  in  the  rods  and  joined  together  by  seg- 
ments. In  my  studies  I  have  repeated  Arnold's  neutral  red  and  methy- 
lene blue  method,  and  I  am  convinced  that  such  a  differentiation  can  be 
obtained.  The  granules  are  stained  red  and  the  segments  blue.  There 
are,  however,  certain  objections.  First  of  all,  neither  dye  is  specific  for 
mitochondria;  secondly,  in  order  to  obtain  a  good  differentiation  of  the 
binding  segments  penetration  has  to  be  prolonged,  and  it  is  well  known 
that  mitochondrial  rods  will  break  up  slowly  into  granules  if  they  are 
exposed  to  changes  in  temperature  or  to  substances  poisonous  to  tissues. 
This  was  shown  by  Lewis  and  Lewis  (16)  in  tissue  cultures,  by  Suzuki 
in  his  carmine  injections,  and  in  various  pathological  conditions  by 
Ophuls(17),  Takaki(18),  Israel  (19),  Burmeister(20),  Landsteiner(21), 
Stork  (22),  Pfister(23),  and  others. 

While  Janus  green  is  considered  a  specific  vital  dye  for  mitochondria, 
it  is  not  a  very  satisfactory  dye  for  the  kidney  as  it  penetrates  very  slowly, 
even  under  the  most  favorable  conditions,  as  on  a  warming  stage.  It  has 
the  tendency  to  bring  out  a  granular  rather  than  a  rodlike  structure,  and 
the  observation  made  here  that  the  granular  structure  was  seen  more 
often  in  the  urea-fed  rats  may  be  the  result  of  various  circumstances.  If 
no  warming  stage  is  used  the  difference  in  temperature  alone  may  be 
sufficient  to  produce  granular  dissolution.  If  a  stage  is  employed  such 
changes  in  osmosis  as  may  be  produced  by  evaporation  may  create  granu- 
lar changes.  Cesa-Bianchi(24)  has  protested  against  the  assumption  of 
pre-existing  granules  on  the  basis  that  unstained  fresh  tissues  have  solid 
rods.    He  maintains  that  the  granular  picture  is  created  by  fixation  or  by 


RESULTS  OF  VITAL  STAINING  STUDIES  113 

the  influence  of  foreign  substances.    It  did  occur  to  me  that  by  polariza- 
tion such  a  question  could  be  settled.    Unfortunately,  at  the  time  of  this 
investigation  this  chance  was  overlooked, 
y/  The  only  result  in  this  part  of  the  investigation  that  can  be  offered 

as  a  fact  is,  that  in  Janus  green  studies  the  tubules  which  were  proven 
by  Leschke(12)  and  Oliver  (13)  to  give  off  urea  also  are  the  ones  that 
have  the  greatest  abundance  of  mitochondria,  and  that  the  tubules  which 
Suzuki  (14)  considered  the  resorption  tubules,  have  no  or  very  little  mito- 
chondrial structure.  But  that  there  is  a  difference  in  vital  staining  reac- 
tion between  the  mitochondria  of  kidneys  after  long-continued  or  acute 
strain  in  urea  excretion,  as  compared  with  those  acting  normally,  cannot 
be  asserted. 

Since  in  some  of  the  first  tissues  studied  vitally  there  were  seen 
spherical,  highly  refractile  bodies  of  various  sizes  apart  from  mitochon- 
dria, Sudan  III  and  scarlet  r.  were  employed  in  the  remaining  tissues 
after  a  Janus  green  penetration  had  been  obtained.  These  round  bodies 
stained  brilliantly  and  instantaneously  with  the  so-called  fat  dyes  which 
did  not  influence  the  green-stained  mitochondria.  The  arrangement  of 
these  bodies  Was  irregular  and  without  any  apparent  similarity  in  neigh- 
boring cells  in  the  entire  group  of  tissues  studied.  That  is  to  say,  they 
were  as  numerous  in  the  controls  as  in  the  urea-fed  rats  of  the  first  and 
second  group.  From  this  I  judge  that  normally  there  is  free  fat,  or  a 
substance  related  to  fat,  in  the  cells  of  the  kidney  of  white  rats.  At  no 
time  did  these  round  bodies  stain  with  Janus  green,  and  this  dye  is  specific 
for  mitochondria.  It  is  obvious  that  these  bodies  are  not  aberrant  physi- 
cal forms  of  mitochondria.  In  the  fixed  sections  similar,  or  perhaps  the 
same,  structures  have  been  seen  by  several  investigators,  and  explanations 
have  been  offered  which  point  out  that  these  bodies  may  be  concerned 
with  secretion.  Heidenhain(25),  Hoeber  and  Koenigsberg(26),  Van  der 
Stricht(27),  and  Gurwitsch(28)  have  described  intracellular  vacuoles  of 
various  positions  and  sizes.  These  vacuoles  were  thought  to  act  as  collec- 
tors of  secretory  materials.  Gross  (29)  and  Hirsch(30)  were  not  able  to 
confirm  the  presence  of  these  vacuoles.  Retzius(31)  observed  these  struc- 
tures but  did  not  think  them  vacuoles.  He  rather  interpreted  them  as  true 
droplets  of  secretion.  In  these  droplets  he  saw  very  fine  granules  con- 
nected by  fine  threads.  At  a  time  when  the  cell  appeared  high  and  the 
brush  border  had  disappeared  these  droplets  were  supposed  to  be  expelled 
into  the  lumen  of  the  tubule. 

In  my  fixed  sections  the  position  and  sizes  of  the  occasional  "vacu- 
oles" observed  seemed  to  correspond  in  location  to  the  fat  bodies  de- 
scribed above.  At  no  time  could  I  find  sufficient  proof  that  in  the  vitally 
stained  tissues  fat  droplets  were  similar  in  arrangement  or  staining  to 
mitochondria.    Nevertheless,  it  may  be  possible  that  these  substances  are 


114  A  CYTOLOGICAL  STUDY  OF  THE  KIDNEY  CELL 

derived  from  mitochondria  as  pointed  out  by  Ophuls(17),  but  if  that  is 
so  it  is  evident  that  some  change  has  occurred  involving  radical  altera- 
tion in  the  staining  affinity  of  the  mitochondrial  substance. 

Results  of  Fixed  Tissue  Studies 

In  studying  the  low-power  picture  the  cortex  stands  out  massively 
on  account  of  the  mitochondrial  richness  of  its  tubules.  This  picture 
varies  in  its  color  intensity  with  the  variations  in  mitochondrial  density 
and  also  with  the  variations  in  fixing  agents.  Nevertheless,  the  color 
coefficient  between  medulla  and  cortex  remains  stationary.  On  viewing 
the  cortex,  even  with  the  lowest  power,  one  can  easily  distinguish  the 
proximal  convoluted  tubules  on  account  of  their  greater  staining  intensity. 
Fig.  1  gives  a  fair  demonstration  of  the  difference  in  color  density  between 
proximal  and  other  tubules  of  the  cortex. 

In  comparing  sections  from  the  four  groups  of  kidneys  differences 
could  always  be  found,  but  further  study  invariably  showed  that  such 
differences  also  existed  within  the  individual  group  and  therefore  could 
not  be  used  in  differentiating  the  groups.  Only  the  study  of  serial  sections 
can  protect  against  unwarranted  conclusions.  As  a  demonstration  I  wish 
to  cite  an  observation  which,  without  serial  study,  would  easily  have  led 
to  faulty  inferences,  if  coincidence  should  have  thrown  the  observations 
mostly  into  one  group  of  tissues. 

In  the  studies  of  the  group  described  by  Dr.  Addis  a  peculiar  picture 
of  divisional  action  of  groups  of  convoluted  tubules  is  observed,  if  one 
may  use  this  term  for  the  sake  of  simplicity  (see  Figs.  2  and  3).  It  is 
at  once  apparent  that  in  some  tissues  entire  groups  of  convoluted  tubules 
with  their  connecting  segments  (Schaltstucke)  stand  out  very  plainly  by 
virtue  of  their  mitochondrial  concentration  in  contrast  to  other  neighbor- 
ing groups,  which  have  less  and  fainter  staining,  but  otherwise  similar 
appearing  mitochondria.  A  casual  observer  might  be  easily  misled  by 
such  a  picture.  In  this  study  it  seemed  at  first  that  such  a  grouping  was 
more  often  seen  in  the  tissues  of  the  rats  which  had  undergone  prolonged 
urea  feeding.  But  "the  study  of  a  large  number  of  sections  from  all  the 
groups  dissipated  this  impression.  All  kidneys  showed  this  peculiarity  in 
some  part  or  other  of  their  structure.  While  this  phenomenon  does  not 
give  any  help  in  differentiating  the  kidneys  of  urea-fed  from  control  rats, 
it  is  of  interest  in  relation  to  Lindemann's(32)  hypothesis  of  a  division 
of  labor  in  the  individual  kidney  in  which  certain  groups  of  tubules  sup- 
posedly are  allowed  to  rest  while  others  are  active.  At  any  rate  it  illus- 
trates the  fallacies  of  drawing  conclusions  from  sections  which  are  not 
representative  of  the  entire  organ. 

The  absence  of  any  appreciable  increase  in  the  weight  of  the  kidneys 
in  Group  I,  which  had  been  fed  urea  over  a  long  period,  precluded  the 
existence  of  any  gross  difference  in  the  number  and  size  of  the  cells  as 


RESULTS  OF  FIXED  TISSUE  STUDIES  115 

compared  with  the  second  group.  Yet,  it  was  thought  possible  that  there 
might  be  a  constant  difference  in  the  minute  structural  arrangement  with- 
in the  cell,  and  a  careful  comparison  was  therefore  made  in  order  to  de- 
termine whether  such  qualitative  distinctions  might  not  be  associated  with 
the  various  degrees  in  urea  feeding,  especially  as  compared  with  the  group 
which  did  not  receive  any  urea  at  all. 

In  studying  the  general  cell  picture  one  finds  that  the  height  of  the 
cell  of  the  proximal  convoluted  tubule  is  fairly  constant,  and  such  varia- 
tions as  exist  seem  to  depend  somewhat  on  the  mitochrondial  content  of 
the  cell.  If  the  content  is  low  the  icell  cupola  is  high  and  the  lumen  is 
very  narrow  (see  Figs.  6  and  7).  This  is  especially  true  when  the  mito- 
chondrial apparatus  is  in  a  state  of  advanced  granular  dissolution  (see 
Fig.  8).  With  the  increase  in  the  numerical  content  of  the  mitochondria 
the  cell  becomes  somewhat  lower.  The  cell  cupola  at  once  becomes  clear 
and  the  plasma  just  at  the  base  becomes  transparent.  The  more  definitely 
the  mitochondrial  apparatus  assumes  the  "batonne"  arrangement  the 
more  apparent  becomes  this  decrease  in  cellular  height  (see  Fig.  11). 
With  this  go  certain  variations  in  configuration  and  width  of  the  lumen. 
When  the  mitochondrial  content  is  low  and  the  plasma  is  dense  through- 
out the  cell  the  lumen  is  narrowest  and  somewhat  stellate  in  character 
(see  Figs.  6  and  7).  As  soon  as  the  granular  structures  become  definite 
in  their  arrangement,  the  lumen  widens  and  only  fine  strands  of  proto- 
plasmic material  bridge  the  lumen  in  an  irregular  fashion  (see  Fig.  12). 

In  comparing  the  cells  of  the  tubular  structure  of  the  kidneys  of  the 
various  groups  it  was  at  once  apparent  that  those  from  animals  fed  urea 
did  not  show  any  evidence  of  a  distinctive  increase  in  the  size  of  the  cells 
of  any  part  of  the  tubular  apparatus.  In  any  kidney  from  any  group  it 
was  possible  to  find  the  lesser  degrees  in  variations  in  the  size  of  individ- 
ual cells  which  have  been  discussed  above  (see  Figs.  9  and  10).  One  may 
therefore  conclude  that  urea  feeding  does  not  stimulate  hypertrophy  of 
the  cell  of  the  kidney  even  if  such  feeding  is  carried  to  the  extreme  by 
extending  it  over  the  life  period  of  the  animal  in  question. 

Also  the  appearance  of  the  brush  border  does  not  offer  any  conclu- 
sive evidence  that  it  is  influenced  in  any  way  by  the  forced  feeding  of 
urea.  I  cannot  agree  with  those  writers  who  think  that  the  variations 
in  the  morphological  appearance  of  the  brush  border  are  indicative  of 
phases  in  the  secretory  process.  By  some  it  is  claimed  that  it  gradually 
rises,  due  to  cellular  pressure,  until  it  is  thrown  off  to  allow  the  secretory 
material  to  enter  the  lumen.  Others  do  not  share  this  view,  and  consider 
the  passage  of  secretion  material  as  a  true  secretion  phenomenon.  Espe- 
cially Sauer(33)  in  his  earlier  studies  opposed  the  former  idea,  since  he 
always  found  a  typically  striated  appearance  of  the  brush  border  at  any 
stage  of  secretion.    Of  late  Kolster(6)  once  more  has  drawn  attention  to 


116  A  CYTOLOGICAL  STUDY  OF  THE  KIDNEY  CELL 

the  brush  border.  He  believes  that  in  what  he  calls  the  "resting  stage" 
(when  the  cells  are  high  and  the  lumen  narrow)  the  brush  border  is  low 
and  possesses  a  very  distinct  striation,  while  in  the  active  stage  the  brush 
border  increases  in  thickness  and  appears  indistinct  in  its  markings. 

Whatever  may  be  the  significance  and  affiliation  of  the  brush  border 
with  other  cell  structures,  it  undoubtedly  is  one  of  the  finest  architectural 
tissue  structures  and  therefore  exposed  to  the  greatest  variations  in  fixing 
artefacts.  In  these  studies  this  was  confirmed  by  the  careful  examination 
of  pieces  of  tissue  where  for  unknown  reasons  penetration  had  lagged. 
Here,  in  sections  cut  deeper  in  the  piece  of  the  tissue,  the  brush  border 
was  severely  damaged  and  often  had  a  "glued-together"  appearance. 
Such  a  condition  may  easily  lead  to  misinterpretations.  And  as  coinci- 
dence occasionally  will  produce  such  an  artefact  in  a  group  of  tubules 
where  the  mitochondrial  content  is  low,  it  is  easy  to  associate  the  two  and 
regard  them,  as  Kolster(6)  does,  as  indicative  of  a  distinct  phase  in  se- 
cretion. I  admit  that  there  are  differences  in  the  thickness  of  the  brush 
border,  and  this  is  regulated  somehow  by  the  condition  of  the  plasma  of 
the  cell  of  the  convoluted  tubule,  since  the  brush  border  appears  clearest 
whenever  the  cell  cupola  is  most  transparent.  This  is  true  of  vitally 
stained  as  well  as  fixed  tissues.  Just  because  it  is  so,  it  may  be  an  optical 
illusion,  because  of  the  fact  that  more  light  is  permitted  to  strike  the 
brush  border  from  all  sides  under  this  condition,  while  it  can  strike  the 
brush  border  only  from  three  sides  when  the  plasma  is  dense,  and  there- 
fore the  striations  may  not  be  seen  as  clearly.  Consequently  the  distinct- 
ness of  the  brush  border  striations  must  vary  normally.  The  actual 
height  of  the  brush  border  varies  very  little  within  the  individual  section 
of  the  proximal  tubule.  In  well  fixed  tissues  I  have  never  seen  a  place 
where  the  brush  border  was  shed  or  being  lifted  off.  I  am  unable  to  pre- 
sent any  photomicrographic  proof  for  this  statement,  as  I  have  not  suc- 
ceeded in  taking  a  picture  which  will  show  the  finer  inner  cell  structure 
clearly  and  at  the  same  time  do  justice  to  the  brush  border.  In  fact,  with 
the  mitochondrial  stains  brush-border  photography  is  almost  impossible. 

Occasionally  one  sees  granular  structures  in  the  brush  border  (see 
Figs.  4,  8,  12).  They  take  the  same  stain  as  do  the  granular  structures 
of  the  cell  itself.  These  granular  bodies,  which  vary  greatly  in  size  and 
which  are  commonly  larger  than  mitochondria  when  there  is  a  dissolu- 
tion of  the  rods,  may  constitute  a  direct  or  indirect  product  of  the  mito- 
chondrial apparatus  and  merely  represent  a  physical  combination  of  more 
than  one  of  the  large  granules  seen  in  the  cell  cupola  in  certain  phases 
in  the  life  cycle  of  the  cell  of  the  convoluted  tubule,  or,  in  fact,  in  any  of 
the  cells  of  the  tubular  structure  in  general  (see  Fig.  8).  Only  occasion- 
ally are  similar  granules  seen  in  the  cell  lumen  or  within  castlike 
formations.    Also,  here  they  do  not  differ  morphologically  from  the  cell 


RESULTS  OF  FIXED  TISSUE  STUDIES  117 

granules.  To  call  them  mitochondrial  secretion  products  offhand  is  open 
to  criticism,  since  we  have  not  accepted  it  as  an  absolute  fact  that  the 
mitochondria  of  the  kidney  enter  into  the  secretory  role. 

When  we  now  compare  the  various  groups  of  the  kidneys  studied  in 
reference  to  the  appearance  of  their  brush  borders,  it  again  must  be  ad- 
mitted that  there  is  no  proof  that  this  structure  is  influenced  in  any  way 
by  urea  feeding.  Such  variations  as  may  be  found  can  be  demonstrated 
within  each  individual  group,  and  therefore  must  be  considered  as  a  nor- 
mal variation  of  the  brush-border  picture. 

In  the  studies  of  the  mitochondrial  apparatus  of  the  kidney  of  the 
white  rat  certain  generalities  have  been  agreed  upon  by  all  investigators. 
Upon  general  survey  one  is  always  impressed  with  the  intense  staining  of 
the  proximal  and  to  a  lesser  degree  of  the  distal  convoluted  tubules.  In 
the  proximal  tubules  the  mitochondrial  apparatus  is  very  distinct  and  con- 
sists of  true  rods  or  of  varying  degrees  of  granular  dissolution  of  these 
rods.  The  mitochondrial  numerical  concentration  is  higher  here  than  in 
other  segments  of  the  tubule.  Also  the  affinity  of  the  individual  granule 
for  certain  stains  is  greater  because  the  granule  is  larger  here  than  in  any 
other  segment  (see  Figs.  4  and  5).  Both  conditions  explain  the  color 
prominence  of  the  proximal  and,  to  a  lesser  degree,  of  the  distal  convo- 
luted tubules.  The  greater  staining  affinity  is  aparently  of  purely  physical 
and  not  chemical  origin,  this  assumption  being  based  on  a  greater  concen- 
tration of  the  mitochondrial  material  in  one  unit.  In  the  medial  and  dis- 
tal parts  of  the  proximal  tubules  the  mitochondrial  structure  is  less  dense 
and  the  granular  dissolution  more  pronounced.  The  descending  limb  of 
the  loop  of  Henle  is  very  poor  in  granules  and  occasionally  appears  free 
from  them,  so  that  it  is  hard  to  recognize  them  with  mitochondrial  stains, 
the  chainlike  or  rodlike  arrangement  of  the  mitochondria,  which  is  so 
typical  of  the  proximal  convoluted  tubules,  is  suggested  again  in  the  mito- 
chondrial arrangement  of  the  ascending  limb  of  Henle's  loop.  But  here 
the  granules  are  very  much  more  scarce,  smaller  and  quite  irregularly 
arranged.  Especially  in  the  cell  cupola  this  irregular  arrangement  is  ap- 
parent and  gives  to  it  a  peculiar,  loose  appearance.  The  distal  convoluted 
tubules  are  similar  to  the  proximal  convoluted  tubules,  except  that  the 
mitochondrial  apparatus  is  less  dense  and  the  cell  is  lower  in  general. 
The  epithelium  of  the  collecting  tubule  is  very  low  and  the  granular  struc- 
tures fine  and  well  scattered  (see  Figs.  13  and  14). 

Studying  on  this  general  basis  the  kidneys  of  the  rats  which  had 
undergone  prolonged  urea  feeding,  and  comparing  the  results  with  those 
of  the  rats  of  the  other  three  groups,  no  essential  difference  in  the  mito- 
chondrial apparatus  can  be  detected.  Also  in  this  group  the  intensely 
staining  mitochondria  of  the  proximal  convoluted  tubules  dominate  the 
picture  (see  Figs.  4,  5,  9,  10,  12).    But  on  careful  study  one  is  at  once 


118  A  CYTOLOGICAL  STUDY  OF  THE  KIDNEY  CELL 

impressed  with  the  want  of  constancy  in  the  appearance  of  the  mitochon- 
dria in  different  or  even  in  the  same  microscopic  sections  from  the  same 
kidney.  This  holds  good  also  in  the  other  groups.  Serial  sections  prove 
that  in  every  kidney  distinct  variations  in  the  structural  picture  of  the 
mitochondria  of  the  same  proximal  convoluted  tubule  are  always  present ; 
that  is  to  say,  the  variations  depend  upon  the  level  at  which  the  section 
is  cut.  There  is  always  a  gradual  transition  in  the  shape  of  the  mito- 
chondrial structure  from  one  segment  of  the  tubule  to  the  other.  Even 
within  the  central  limit  of  the  segments  studied  such  variations  are  pres- 
ent. Then  again  neighboring  segments  of  the  same  type  but  of  different 
tubular  units  may  vary  distinctly  in  the  structural  arrangement  of  their 
mitochondria  as  to  size,  number,  and  shape.  But  such  variations  cannot 
be  the  result  of  urea  feeding,  since  they  are  also  found  in  the  kidneys 
of  the  rats  that  were  not  fed  any  urea  at  all.  There  are  about  as  many 
variations  as  there  are  kidneys  studied,  and  I  am  afraid  one  could  carry 
the  descriptions  of  the  various  combinations  of  these  on  to  the  infinite. 
True  enough,  on  a  general  quick  survey  of  the  sections  of  the  group 
that  received  no  urea,  the  picture  of  the  kidney  of  the  white  rat  described 
for  the  normal,  holds  good,  but  when  it  comes  to  comparing  even  neigh- 
boring sections  of  the  same  tubular  group,  variations  are  also  found  here 
at  once.  For  instance,  there  is  a  distinct  difference  in  the  granular  disso- 
lution in  Figs.  8  and  12,  which  are  sections  from  the  controls  of  the  first 
group.  But  the  same  is  true  of  the  kidneys  of  the  rats  fed  urea  over  a 
long  period  (see  Figs.  6,  9,  10).  Then  again  the  dogmatic  picture  which 
has  been  described  for  the  normal  is  represented  by  Fig.  11,  but  it  is  a 
picture  of  the  proximal  convoluted  tubule  from  a  kidney  exposed  to  a  long 
urea  strain.  Here  the  beautiful  "batonne"  arrangement  is  very  apparent 
and  dominates  the  optical  impression  received.  These  instances  merely 
illustrate  the  point  raised. 

In  comparing  the  individual  mitochondrial  granules  of  the  various 
groups  no  hypertrophy  was  observed  in  those  where  urea  had  been  fed, 
and  when  compared  to  controls  from  the  first  and  second  group  no  nu- 
merical increase  could  be  established.  It  is  impossible  to  segregate  the 
various  groups  of  rats  by  the  appearance  of  their  mitochondrial  apparatus. 
Granular  dissolution  or  breaking  up  of  the  solid  rods  is  not  any  more  a 
characteristic  of  the  urea  kidneys  than  of  the  control  kidneys  (compare 
Figs.  6  and  7,  9  and  12).  Nor  is  the  size  of  the  granules  produced  dur- 
ing dissolution  any  indication  that  the  mitochondrial  apparatus  of  the 
proximal  convoluted  tubules  is  in  any  way  affected  by  increased  urea 
'  secretion,  since  the  same  picture  is  found  in  controls  that  did  not  receive 
any  urea  whatsoever.  If  we  pay  attention  to  the  qualitative  difference  in 
staining1  it  can  be  stated  that  there  is  none  except  for  the  usual  normal 
variation. 


RESULTS  OF  FIXED  TISSUE  STUDIES  119 

The  picture  of  the  mitochondrial  structure  of  the  other  tubular'  seg- 
ments corresponds  in  general  to  those  described  for  the  normal  with  cer- 
tain small  variations  under  ordinary  conditions.  The  glomeruli  and  their 
capsules  are  practically  free  from  mitochondria  in  the  kidney  under  normal 
activity,  and  urea  feeding  does  not  bring  on  an  increase  (see  Figs.  15  and 
16).  There  are  no  indications  anywhere  in  the  tissues  that  pathological 
changes  had  been  produced  by  urea  feeding. 

A  number  of  observers  have  ascribed  a  secretory  role  to  the  mito- 
chondria of  the  kidney,  such  conclusion  being  based  on  animal  experi- 
mentation. For  instance,  during  hibernation  Ferrata(34),  Disse(35),  and 
Monti (36)  found  the  following  changes  from  the  normal  under  ordinary 
circumstances :  the  lumina  of  the  convoluted  tubules  are  narrow  and  the 
epithelium  is  not  sharply  outlined  but  markedly  granular.  Near  the  base 
the  granules  assume  a  regular  chainlike  arrangement,  while  the  inner  and 
less  dense  part  of  the  cell  is  traversed  by  fine  threads.  When  secretion 
sets  in,  this  latter  part  of  the  cell  becomes  quite  transparent,  bulges  toward 
the  lumen  and  granules  replace  the  threads.  This  appearance  was  con- 
sidered a  storage  of  granules  supposedly  characteristic  for  a  prophase  of 
secretion. 

Also  Takaki(18),  working  with  starvation  and  dry  feeding,  and 
Kolster(6),  using  similar  methods  together  with  sodium  chloride  injec- 
tions, concluded  that  mitochondria  have  a  definite  place  in  the  secretory 
process  of  the  kidney.  Their  investigations  tend  to  divide  the  stages  of 
secretion  according  to  the  appearance  and  arrangement  of  mitochondria. 
Their  findings  have  been  confirmed  by  Schultze(37).  It  is  of  greatest 
importance  that  Regaud(38),  after  most  exhaustive  studies,  concluded 
that  all  the  various  types  of  granules,  rods,  and  filaments  described  under 
different  names  belong  to  the  same  group,  namely,  mitochondria,  and  that 
they  varied  in  their  appearance  according  to  a  number  of  conditions 
among  which  were  changes  in  the  secretory  activity  of  the  cell.  It  was 
Simon (39),  Retterer(40),  and  Henschen(41),  and  to  some  degree 
Dumas (42),  Mayer  and  Rathery  (43),  who  substantiated  Regaud's  con- 
clusion by  further  experimental  work.  Nevertheless,  the  changes  men- 
tioned have  been  thought  to  be  artefacts  by  Prenant(44),  Policard(45), 
Levi (46),  and  Ciaccio(47). 

I  cannot  agree  with  the  latter,  since  I  feel  certain  that  the  mito- 
chondrial play  seen  in  these  studies,  and  of  which  photomicrographs  are 
presented  here,  is  not  the  result  of  variations  in  fixation.  It  is  true  that 
different  fixations  (fixing  agents)  of  pieces  from  the  same  tissue  will  show 
slight  differences  in  the  appearance  of  the  individual  mitochondrial  unit, 
but  aside  from  this  the  variations  observed  within  one  piece  are  constant 
for  that  fixation,  and  correspond  to  the  variations  in  pieces  fixed  differ- 


120  A  CYTOLOGICAL  STUDY  OF  THE  KIDNEY  CELL 

ently,  after  one  has  allowed  for  the  deduction  of  such  changes  as  are 
produced  by  the  fixing  agents  generally. 

Leschke,  and  later,  Oliver,  found  that  urea  was  secreted  in  the  prox- 
imal convoluted  tubules.  The  mitochondrial  concentration  is  highest  in 
this  part  of  the  tubular  apparatus,  and  the  mitochondrial  changes,  which 
by  many  have  been  thought  to  be  of  secretory  importance,  are  also  here 
most  pronounced.  It  is,  therefore,  only  natural  to  attempt  to  link  together 
these  observations.  But  the  studies  cited  here  lead  me  to  believe  that  urea 
feeding  of  various  degrees  does  not  influence  beyond  normal  variations 
the  physical  appearance  of  the  mitochondrial  apparatus  of  the  kidney  of 
the  white  rat  in  general  and  of  its  proximal  convoluted  tubules  in  par- 
ticular. 

Summary 

The  result  of  an  anatomical  comparison  between  the  kidneys  of  white 
rats  on  a  mixed  diet,  calling  for  no  unusual  renal  activity,  and  the  kidneys 
of  rats  which  for  prolonged  or  brief  periods  of  time  had  undergone  a 
markedly  abnormal  strain  in  the  excretion  of  large  amounts  of  urea,  may 
be  summed  up  as  follows : 

1.  Prolonged  urea  feeding  led  to  no  increase  in  the  size  of  the  cells 
of  any  part  of  the  glomerulo-tubular  apparatus. 

2.  Neither  a  prolonged  nor  a  brief  increase  in  urea  excretion  had  any 
observable  effect  on  the  appearance  of  the  brush  border. 

3.  A  careful  study  of  the  mitochondrial  apparatus  of  the  kidney 
failed  to  show  that  variations  in  the  rate  of  urea  excretion  during  life 
were  associated  with  any  change  in  the  number,  size  or  position  of  the 
mitochondria. 

I  take  occasion  to  express  my  sincere  appreciation  to  Dr.  T.  Addis 
for  his  most  helpful  suggestions  in  editing  this  paper,  to  Miss  Ella  Wing 
for  her  splendid  help  in  the  technical  work,  and  to  Dr.  F.  E.  Blaisdell  for 
his  kind  advice  in  photographic  difficulties. 

Stanford  University  Hospital,  San  Francisco 


BIBLIOGRAPHY  121 


BIBLIOGRAPHY 

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2.  Robertson,  T.  B.,  and  Ray,  L.  A. :   Journal  of  Biol.  Chent.,  24,  347,  1916. 

3.  Osborne,  T.  B.,'and  Mendel,  L.  B.:  Journal  of  Biol.  Chem.,  17,  401,  1914. 

4.  Bensley,  R.  R.:   Amer.  Jour.  Anat.,  12,  297,  1911. 

5.  Regaud,  C. :   Arch.  d'Anat.  micr.,  11,  291,  1910. 

6.  Kolster,  R. :   Ziegler's  Beitrdge,  51,  209,  1911. 

7.  Altmann,  R. :    "Die  Elementarorganismen,  etc.,"  p.  27.   Leipzig,  Veit  Co.,  1890. 

8.  Cowdry,  E.  V. :    Contributions  to  Embryology,  Carnegie  Institution,  Washing- 

ton.   No.  11,  p.  27,  1916. 

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10.  Arnold,  J. :    Virchovfs  Archiv.,  169,  1,  1902. 

11.  Heidenhain,  R. :   Arch.  /,  micr.,  Anat.,  10,  1,  1874. 

12.  Leschke,  Erich : 

(1)  l'ter  Kongress  fur  innere  Medizin,  Wiesbaden,  1914,  p.  635. 

(2)  Zeitschr.  f.  Klin,  Medizin,  81,  14,  1914-15. 

13.  Oliver,  Jean :   J  our,  of  Experimental  Medicine,  23,  301,  1916. 

14.  Suzuki,  T. :  "Zur  Mbrphologie  d.  Nierensekretion."    Jena,  1912,  Gustav  Fischer. 

15.  Arnold,  J. :  "Ueber  Plasmastrukturen."    Jena,  1914,  Gustav  Fischer. 

16.  Lewis,  M.  R.,  and  W.  H.:   Am,  Jour,,  of  Anat.,  17,  339,  1915. 

17.  Ophiils,  W.:   Proc.  Soc.  Exp.  Biol,  4,  135,  1907. 

18.  Takaki,  K. :   Arch  f,  mikr.  Anat.,  70,  245,  1907. 

19.  Israel,  O. :    Virchowfs  Arch.,  123,  310,  1891. 

20.  Burmeister,  Th. :    Virchow's  Arch.,  137,  405,  1894. 

21.  Landsteiner,  K. : 

(1)  Wiener  klin.  Wochschft.,  41,  1904. 

(2)  Ziegler's  Beitraege,  33,  157,  1903. 

22.  Stork,  O.:    Sitzbr.  d.  k.  Akad.  d.  Wissenschaften,  Wien.,  115,  Abt.  3,  1906. 

23.  Pfister,  M. :   Ziegler's  Beitraege,  Arnold's  Festschrift,  37,  Suppl.  7,  p.  525,  1905. 

24.  Cesa-Bianchi,  D.:    Intern.  Mtschrft.  f.  Anat.  u.  Phys.,  27,  89,  1910. 

25.  Heidenhain,  M. :    "Plasma  und  Zelle,"  Jena,  G.  Fischer,  1911. 

26.  Hoeber,  R.,  and  Koenigsberg,  A. :   Pflueger's  Arch.,  68,  323,  1905. 

27.  Van  der  Stricht,  O. :    Annal.  d-  I.  Soc.  d.  Med.  Gent.,  1892,  Reprint. 

28.  Gurwitsch,  A.:   Pfliiger's  Arch.,  91,  71,  1902. 

29.  Gross,  W. :   Ziegler's  Beitr.,  51,  528,  1911. 

30.  Hirsch,  C. :   Anat.  Hefte,  41,  131,  1910. 

31.  Retzius,  G. :    Biologische  Untersuchungen,  17,  53,  1911. 

32.  Lindemann,  W. :   Ergebn.  d.  Phys,,  14,  643,  1914. 

33.  Sauer,  H.:   At*ch.  f.  mikr.  Anat.,  46,  109,  1895. 


122  A  CYTOLOGICAL  STUDY  OF  THE  KIDNEY  CELL 

34.  Ferfrata,  A. :    Arch.  ital.  Anat.  e  embr.,  4,  505,   1905   (see  Schwalbe's  Jahres- 

berichte  for  1905,  p.  505). 

35.  Disse,  J. :  Anat.  Hefte,  2,  143,  1892. 

36.  Monti,  R.,  and  A.:    Mem.  let.  ital.  d.  Istitut.  Lomb.  Pavia,  14,  82,  1900. 

37.  Schultze,  O.:   Anal  Ariz.,  38,  257,  1911. 

38.  Regaud,  CI. : 

(1)  C.  R.  Soc.  Biol.,  64,  1145,  1908. 

(2)  C.  R.  Soc.  Biol,  65,  206,  1908. 

39.  Simon,  Ch. :   Compt.  Rend.  Soc.  Biol,  Paris,  50,  443,  1898. 

40.  Retterer,  E. :   Compt.  Rend.  Sofc,  Biol,  Paris,  60,  560  and  611,  1906. 

41.  Henschen,  F.:  Anat.  Hefte,  26,  575,  1904. 

42.  Ribadeau-Dumas,  L. :    Compt.  Rend.  Soc.  Biol,  Paris,  54,  484,  1902. 

43.  Mayer,  A.,  and  Rathery,  F. :  /.  d.  VAnai.  et  de  Phys.,  45,  321,  1909. 

44.  Prenant,  A. :   Compt.  Rend.  Soc.  Biol,  Paris,  59,  218,  1905. 

45.  Policard,  J.:  "Rev.  generale  d'histolog.,"  p.  431,  Masson,  1908. 

46.  Levi,  G.:  Arch,  ital  di  Anat.  e  di  Embr-,  10,  168,  1911. 

47.  Ciaccio,  C. :   Centralbl  f.  allg.  Path.,  55,  131,  1913. 


explanation  of  plates  123 

Explanation  of  Plates 

Technical  Remarks:  All  photographs  were  taken  with  a  Zeiss  micro- 
scope on  Cramer  isochromatic  plates.  For  illumination  a  4-5  amp.  Leitz 
arc-light  filtered  with  a  single  potassium  bichromate  (Orth)  screen  was 
used.  The  ocular  used  for  all  pictures  is  No.  4,  the  objective  for  "low 
power"  is  No.  3,  for  "high  power"  No.  6,  and  for  "oil-immersion"  No. 
1/12  apochromatic.  The  difference  in  size  of  corresponding  structures  in 
different  pictures  photographed  with  the  same  microscopical  magnification 
is  due  to  slight  differences  in  the  length  of  the  bellows  of  the  camera, 
which  has  to  be  varied  according  to  various  circumstances.  For  printing 
Azo  "E  and  F  hard"  were  used. 

All  preparations  reproduced  here  were  fixed  in  either  Regaud's  or 
Bensley's  formalin  bichromate  mixtures,  and  stained  with  a  modified 
"Altmann  anilin  fuchsin,"  in  which  the  destaining  of  the  plasma  with 
picric  acid  had  been  carried  over  the  usual  point.  This  assures  a  high 
contrast  for  mitochondria.  Only  Figure  2  is  from  a  copper-chrome-hema- 
toxylin  preparation. 

Figure  1,  low  power,  urea  fed  just  before  the  death.  The  proximal 
convoluted  tubules  stand  out  by  virtue  of  the  greater  density  of  the  mito- 
chondrial content.    This  is  the  typical  low  power  picture. 

Figure  2,  low  power,  urea  fed  just  before  the  death,  and  Figure  3, 
low  power,  urea  fed  over  a  long  period.  In  contradistinction  to  Figure  1, 
Figures  2  and  3  show  a  distinct  localized  staining  reaction  of  certain 
tubular  groups,  while  the  neighboring  and  otherwise  same  structures  re- 
main faint  in  outline.  In  the  left-hand  corner  of  Figure  3  the  usual  pic- 
ture is  approached  again.  The  localized  staining  reaction  is  due  to  the 
greater  mitochondrial  concentration  in  this  region  and  occurs  in  any 
kidney  under  ordinary  circumstances.  Figure  3  is  somewhat  obscured 
by  faulty  illumination. 

Figure  I,  high  power,  urea  fed  just  before  death,  and  Figure  5,  high 
power,  no  urea  fed,  represent  the  usual  dogmatic  picture  of  the  mito- 
chondrial distribution  in  the  kidney  of  the  white  rat  under  ordinary 
circumstances.  That  one  dose  of  urea  has  no  influence  on  the  mito- 
chondrial apparatus  is  seen  in  Figure  1  by  the  unchanged  appearance  and 
distribution  of  the  granules.  The  proximal  convoluted  tubules  stand  out 
very  distinctly.  The  lumen  is  narrow.  The  brush  border  cannot  be  dis- 
tinguished, for  reasons  explained  elsewhere.  The  distal  convoluted 
tubules,  connecting  segments  (Schaltstuck)  and  the  limbs  of  Henle's  loop 
are  distinguished  easily.  The  descending  limb  of  the  latter  is  free  from 
mitochondria.  The  distal  convoluted  tubules  (segment  intermediaire. 
Schweigger-Seidel's  Zwischenstuck)  are  recognized  by  the  wider  lumen 


124  A  CYTOLOGICAL  STUDY  OF  THE   KIDNEY  CELL 

and  the  fewer  mitochondria.  A.  Glomerulus.  B.  Proximal  convoluted 
tubule.  C.  Distal  convoluted  tubule.  D.  Descending  limb  of  H.  L. 
E.  Ascending"  limb  of  H.  L.     F.  Connecting  segments. 

Figure  6,  high  power,  urea  fed  during  entire  length  of  life,  and  Figure 
7,  oil  immersion,  no  urea  fed  at  all,  show  the  end  stage  in  granular  disso- 
lution. There  is  no  difference  in  size,  appearance  and  arrangement  of  the 
granules.  The  lumina  of  the  tubules  (proximal)  are  narrow  and  the 
cells  are  high  in  either  urea-fed  and  regular-diet  animal.  In  the  latter  the 
chainlike  arrangement  of  the  granules  is  still  preserved.  This  configura- 
tion has  been  described  as  the  end  stage  of  secretion. 

Figure  8,  high  power,  urea  fed  just  before  death,  and  Figure  9,  oil 
immersion,  urea  fed  during  the  entire  life,  apparently  represent  distinctive 
and  different  types  of  granular  dissolution.  But  comparing  Figure  8 
with  Figure  12,  it  is  at  once  apparent  that  these  differences  are  only  indi- 
vidual variations  of  granular  dissolution  in  the  same  kidney.  Both  these 
pictures  are  from  the  same  kidney  but  from  different  levels.  In  Figure  8 
a  clumping  of  mitochondria  into  larger  bodies  staining  like  mitochondria 
is  distinctly  seen.  In  Figure  8a  this  has  been  brought  out  still  more 
markedly  by  lengthening  the  bellows  of  the  camera. 

Figure  10,  oil  immersion,  urea  fed  for  fourteen  days,  shows  the  usual 
variations  in  the  height  of  the  cells  and  the  difference  in  the  mitochondrial 
density  within  one  tubule.  There  is  an  early  granular  dissolution  present, 
but  the  rodlike  arrangement  is  still  preserved ;  the  picture  is  not  different 
from  the  normal. 

Figure  11,  high  power,  urea  fed  during  the  entire  length  of  life,  shows 
the  typical  batonne  arrangement  described  for  the  normal  and  which  is 
not  disturbed  by  intense  urea  feeding. 

Figure  12,  high  power,  urea  fed  just  before  death,  shows  the  early 
granular  dissolution  which  occurs  normally.  The  brush  border  is  just 
faintly  visible,  not  changed  and  contains  a  few  fine  granules  with  a  mito- 
chondrial staining  reaction. 

Figure  13,  oil  immersion,  urea  fed  during  entire  lifetime,  and  Figure 
11,  oil  immersion,  no  urea  fed  at  all,  show  primarily  that  the  collecting 
segments  of  the  tubular  apparatus  is  partially  free  from  mitochondria  and 
that  the  few  mitochondria  which  are  present  are  not  altered  in  any  way  or 
increased  in  number  by  prolonged  urea  feeding. 

Figure  15,  oil  immersion,  prolonged  urea  feeding,  and  Figure  16,  oil 
immersion,  no  urea  feeding,  show  that  the  glomerular  apparatus  is  not 
influenced  by  a  severe  strain  such  as  is  produced  by  prolonged  urea 
feeding;. 


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